I. N-(phosphonacetyl)-L-aspartate (PALA, a transition state analog inhibitor of aspartate transcarbamylase, blocks de novo pyrimidine biosynthesis in mammalian cells in culture. Cells that are resistant to killing concentrations of PALA have greatly increased levels of aspartate transcarbamylase and carbamyl-P synthetase. We want to investigate the causes of these increases. We will attempt to purify the complex of early pyrimidine enzymes from overproducing mutants by affinity chromatography, prepare antibodies to the purified components of the complex and use these antibodies to precipitate specific polysomes making each type of polypeptide in order to estimate the amount of specific RNA being translated. Other experiments will be done to evaluate possible increases in DNA coding for these enzymes, differential translational control and differential control of enzyme degradation. II. Two new antigens have been identified in a line of SV40-transformed hamster cells. Y antigen is SV40-specific and is found in the nucleus but is distinct from the well-known T antigen which has similar properties. Attempts will be made to develop high-titer specific antisera to Y and T so that they can be purified and studied with respect to structure and function. X antigen is associated with messenger ribonucelo-proteins, both free and in polysomes. We want to determine the type of RNA to which X is bound, purify X, and determine its role in vivo by recombining it with RNA and studying the consequences in vitro. III. The protein components of messenger ribonucleoproteins isolated from specific polysomes for early pyrimidine pathway enzymes will be compared with each other, with X antigen and with proteins from total polysome-bound mRNPs. Reconstitution experiments with pure proteins and RNAs will be attempted in order to define stoichiometry relationships and functions of the proteins in vitro.